PROJECT SUMMARY Cellular processes such as signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions in crowded cellular environments. Aberrant protein-protein interactions are frequently implicated in debilitating or fatal diseases such as diabetes, neurodegenerative diseases, and cancer. Established structural and cell biological techniques are mostly limited to dissecting the function of stable protein complexes, and do not investigate emergent behavior stemming from multiple transient interactions. To address this challenge, we use DNA nanotechnology scaffolds to pattern macromolecules in vitro and a novel genetically encoded ER/K linker to probe and modulate protein interactions in live cells. Together, we leverage these technologies to dissect the molecular mechanisms of multiplicity in G protein- coupled receptor (GPCR) signaling and biophysical regulation of unconventional myosin function in cells. We have successfully investigated two distinct aspects of GPCR signaling specificity in cells using biosensors engineered by linking GPCR and G protein elements with an ER/K linker. First, we hypothesize that ligands stabilize GPCR conformational sub-states that selectively interface with one or more G? C-termini, to tune ligand efficacy and potency for downstream pathways. Our goal is to use a combination of GPCR biosensors and multi-scale molecular dynamics simulations to define hot-spot residues, structural motifs, and allosteric pathways in both the GPCR and G protein that drive signaling specificity. Second, our research has advanced a role for concurrent and sequential interactions between GPCR and effectors on signaling specificity. Our goal is to combine GPCR biosensors and traditional pharmacology approaches to dissect the synergistic effects of G proteins on GPCR signaling. Together, our research provides insights into GPCR signaling specificity that can be leveraged in structure-based drug discovery efforts to identify functionally selective GPCR ligands. Unconventional myosins are essential in numerous cellular processes including membrane traffic, contractility, and cell division. Defining the motile function of myosins in cells is challenged by the myriad geometries of both actin networks and cargo, paired with a diversity of motor-cargo interfaces. We use cargo-mimetic DNA nanotechnology scaffolds combined with computational modeling to successfully dissect the mechanical coordination in myosin ensembles. We will use these approaches to gain insight into the biophysical regulation of myosin function through the motor-cargo interface. We hypothesize that cargo interfaces act as molecular modules that tune myosin function to match the functional requirements of individual cellular processes. Our goal is to dissect myosin regulation through interactions with distinct cargo adaptors, Rab GTPases, and cell- derived cargo complexes. Our research will advance our understanding of emergent myosin function in cells, while providing a broad theoretical framework for the cargo-mediated regulation of cytoskeletal motors.